Generation of physical map contig-specific sequences

Abstract

Rapid advances of the next-generation sequencing technologies have allowed whole genome sequencing of many species. However, with the current sequencing technologies, the whole genome sequence assemblies often fall in short in one of the four quality measurements: accuracy, contiguity, connectivity, and completeness. In particular, small-sized contigs and scaffolds limit the applicability of whole genome sequences for genetic analysis. To enhance the quality of whole genome sequence assemblies, particularly the scaffolding capabilities, additional genomic resources are required. Among these, sequences derived from known physical locations offer great powers for scaffolding. In this mini-review, we will describe the principles, procedures and applications of physical-map-derived sequences, with the focus on physical map contig-specific sequences.

Keywords: BAC end sequences; assembly; physical map contig-specific sequences; scaffolding; whole genome sequencing.

FIGURE 1

(A) A flow chart illustrating the physical map contig-specific fragment preparation. The BAC clones from each physical map contig were selected, using a minimal tilling path, and then the BAC DNA from each physical map contig were pooled for digestion with two restriction endonucleases. In-house designed adaptors were then ligated to the digestion products, followed by amplification using in-house designed primers. All of the PCR products were pooled together and sequenced (Jiang et al., 2013). (B) Decoding based on the adaptors and primers. Each PCR product was attached to a physical map contig-specific tag at both ends. The tag representing the physical map contig ID contains five parts: ① represents the shields DNA, ② represents the specific barcoded sequencing in the primer (Pj), ③ represents the common/complementary sequences between the adaptor and the PCR primer, ④ represents the specific barcoded sequences in the adaptor (Ai), and ⑤ represents the overhangs proximal to the fragments for ligation (Jiang et al., 2013)

FIGURE 2

Comparison of the anchoring power of only BESs versus BESs plus physical map contig-specific sequences (PMCSSs). The red bar on the BAC clone represents the BESs, while the yellow bar represents the PMCSSs.